Spermatogenesis is a complex process of differentiation that is dependent upon paracrine interactions among the cellular elements in the testis. The different cells (myoid, germ, Leydig, Sertoli) certainly do not function in isolation, but there is a coordinated combination of events that collectively permit spermatogenesis. Our long term objective is to achieve a better understanding of the factors controlling germ cell differentiation. Our research will emphasize the role of the Sertoli cell in the spermatogenic process and the interdependence of the cellular elements within the testis. We recently developed a novel dual compartment (bicameral) culture system which mimics more closely cells growing in vivo. In this part of the Program Project, in the first specific aim, we will continue our experiments to characterize the Sertoli cells in bicameral culture and, in particular, attempt to prove that Sertoli cells growing in this culture chamber are more physiological than cells in conventional culture. Then, in the second aim, we will examine the effects of other testicular cells (myoid/Leydig/germ), individually, on Sertoli cell differentiation and function in the bicameral chamber. The bicameral cell culture chamber has led to many novel experiments in the testis, including the ability to recombine simultaneously the cellular elements of the testis in culture in a manner which is analogous to their position in vivo. After we understand clearly the effects of the individual cell types on the Sertoli cells, we will then be in an excellent position to place all the above cellular elements of the testis in the bicameral chamber, simultaneously. Sertoli cells will be grown on top of extracellular matrix-coated Millipore filters; myoid cells will be placed on the undersurface of the filters, or on top of the filters with the Sertoli cells; Leydig cells will be cultured in the basal chamber; different classes of germ cells will be placed on top of the filter with the Sertoli cells. With these experiments we may be able to achieve a greater degree of germ cell differentiation in culture than has been previously possible, especially because of the newly developed bicameral culture chamber. We will also examine Sertoli cell differentiation and function in the presence of these other three cellular elements. Finally, we have recently demonstrated that the Sertoli cells produce a mitogenic protein which modulates Leydig cell division. We will attempt to determine whether this factor plays a direct role in germ cell mitoses and/or differentiation. The experiments outlined in this proposal should provide new data on the basic biology of the Sertoli cell and its role in spermatogenesis and on the interdependence of the cellular elements in the testis. This approach could well have significance in the development of future male contraceptives.